CN103045727A - SNP (Single Nucleotide Polymorphism) marker related with Chinese Holstein cow milk production property and somatic cell score and application thereof - Google Patents
SNP (Single Nucleotide Polymorphism) marker related with Chinese Holstein cow milk production property and somatic cell score and application thereof Download PDFInfo
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Abstract
The invention relates to an SNP (Single Nucleotide Polymorphism) marker related with a Chinese Holstein cow milk production property and a somatic cell score and an application thereof. A site of the SNP marker is located on a Chinese Holstein cow HAL (Histidine Ammonia-lyase) gene, such as a 197bp site of a sequence shown as SEQ ID NO.1; a basic group of the site is G or A; the content of lactoprotein of the Chinese Holstein cow with the basic group G is obviously higher than that of the Chinese Holstein cow with the basic group A; and the somatic cell score of the former is obviously lower than the latter. The invention further provides a primer for detecting the SNP marker and a kit containing the primer. The invention further provides the application of the SNP marker to a Chinese Holstein cow advantageous variety with high identification lactoprotein content and low somatic cell score. The invention further provides an application of the SNP marker to assistant breeding in a Chinese Holstein cow molecular marker. The SNP marker disclosed by the invention provides scientific basis for marker auxiliary selection of the Chinese Holstein cow milk production property and the somatic cell score.
Description
Technical field
The present invention relates to the domestic animal technical field of molecular biology, be specifically related to a kind of SNP mark and the application thereof relevant with china holstein cows milk production trait and somatocyte scoring.
Background technology
Development of dairy industry is the important symbol of a national modernization of livestock farming.In recent years, through milk cow industry worker's unremitting effort, the development of China's milk cow industry was rapid, for promoting increasing peasant income, improved the physique of the nation people, optimized the structure of agriculture significant.The occupancy volume per person is few, the milk cow single rate is low, improve traditional Development patterns of the total amount of giving milk to expand milk cow quantity, is still the subject matter that the China milk industry development faces.Therefore, cultivate high yield cow colony, milk cattle cultivating has become the key of China's Dairy Industry to quantity and the transformation that lays equal stress on both quality and quantity.Only have the hereditary level that promotes China milk cow colony, improve the milk cow health situation, improve the cow producing milk level, just can make China's milk cow industry realize sustainable and healthy development.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) be a class genetic marker that was proposed by the human genome research centre scholar Lander of Massachusetts Institute Technology in 1996, mainly refer on genomic level by the caused dna sequence polymorphism of the variation of single core thuja acid.The polymorphism that SNP shows only relates to the variation of single base, and manifestation has conversion, transversion, insertion and disappearance etc.
Order-checking, single strand conformation polymorphism polymerase chain reaction (PCR-single-strandconformation polymorphism, PCR-SSCP), the technology such as restriction fragment length polymorphism polymerase chain reaction (PCR-restriction fragment lengthpolymorphism, PCR-RFLP) and flight time mass spectrum can realize the detection of SNP.SNP has been widely used in the assignment of genes gene mapping, clone, genetic breeding and multifarious research as new genetic marker.
Flight time mass spectrum (Matrix-Assisted Laser Desorption/Ionization Timeof Flight, MALDI-TOF) is the detection technique that a kind of accuracy is high, handiness is strong, flux is large, sense cycle is short.Its principle is to utilize the principle that the flight time of sample molecule in electric field be directly proportional with the specific charge of molecule, the flight time by the test sample molecule, record molecular weight, and realize distinguishing, differentiating the purpose of material.Molecular weight there are differences between two allelotrope in SNP site, therefore uses this technology can realize gene type.Flight time mass spectrum (MALDI-TOF) platform has been widely used in biological technical field, has statistics to show that the checking SNP above 95% can both use this platform to analyze and study.
Molecular breeding, i.e. molecular marker assisted selection breeding refers to utilize dna molecular marker that breeding material is selected, and comprehensive improvement livestock and poultry important economical trait is the breeding method that traditional genetic breeding and modern molecular biology organically combine.Molecular breeding is that cattle breeding opens up a new way, and along with the development of modern biotechnology, the effect of molecule marker in cattle breeding will become increasingly conspicuous.In the breeding of milk cow, people wish by to closely related with milk production trait, and with the selection of the closely linked dna marker of quantitative character, realizing early stage seed selection and to improve the target of breeding accuracy, thereby obtain larger genetic progress.
Histidine ammonialyase (Histidine Ammonia-lyase, HAL) is a kind of cytosol enzyme, the first step of catalysis Histidine metabolic process, and namely the non-oxide deamination of L-Histidine forms trans Uregit.The Histidine ammonialyase is the first step rate-limiting enzyme of catalysis Mammals Histidine metabolism, and the variation of HAL gene polynorphisms may change the catalytic efficiency of Histidine ammonialyase, affects Histidine metabolism in the animal body.In china holstein cows colony, carry out whole-genome association, find that the impact of HAL gene pairs milk yield and Milk protein yield reaches conspicuous level.The variation of HAL gene polynorphisms may affect digesting and assimilating of the interior Histidine of Contents in Cows, thereby affects the milk production traits such as its milk yield, Milk protein yield.
Summary of the invention
The purpose of this invention is to provide a kind of SNP mark and the application thereof relevant with china holstein cows milk production trait and somatocyte scoring.
The purpose of this invention is to provide a kind of nucleic acid fragment for detection of china holstein cows milk production trait and somatocyte scoring, the sequence of this nucleic acid fragment is shown in SEQ ID NO.1, and the base of 197bp site is G or A.
The purpose of this invention is to provide a kind of SNP mark relevant with china holstein cows milk production trait and somatocyte scoring, described SNP mark is positioned at the 197bp site of china holstein cows HAL gene sequence shown in SEQ ID NO.1, in SEQ ID NO.1 sequence, represent this site, place with n, and the base in site is G or A herein.197bp site base is that the milk protein content of the china holstein cows of G is significantly higher than herein that base is the china holstein cows of A, and the former significantly is lower than the latter and somatocyte is marked.
Another object of the present invention provides the primer for detection of the SNP mark relevant with china holstein cows milk production trait and somatocyte scoring.
Primer for detection of described SNP mark of the present invention comprises forward primer F:5 ’ – GGCAACTACCTGAACCAA-3 ' and reverse primer R:5 ’ – CACCACCCTGTCAATCAA-3 ', respectively shown in SEQ ID NO.2 and SEQ IDNO.3.
Another object of the present invention provides the test kit for detection of the SNP mark relevant with china holstein cows milk production trait and somatocyte scoring, and this test kit contains aforementioned positive primers F and reverse primer R.
Described test kit comprises dNTPs, Taq archaeal dna polymerase, Mg
2+With the PCR reaction buffer.
Preferably, described test kit also comprises standard positive template.
Another object of the present invention provides described SNP and is marked at the application of identifying in the china holstein cows Dominant variety that Milk protein yield is high, the somatocyte scoring is low.Described being applied as utilizes single nucleotide polymorphism to detect china holstein cows milk production trait and somatocyte scoring.
Described SNP is marked at the application of the china holstein cows Dominant variety of identifying that Milk protein yield is high, the somatocyte scoring is low, may further comprise the steps:
(1) genomic dna of extraction china holstein cows to be measured;
(2) take the genomic dna of china holstein cows to be measured as masterplate, utilize primers F and R, amplify china holstein cows HAL gene 307bp fragment by the PCR reaction, wherein,
F：5’–GGCAACTACCTGAACCAA-3’
R：5’–CACCACCCTGTCAATCAA-3’；
(3) detect pcr amplification product, if the base at 197bp place is G in the amplified production sequence, then china holstein cows to be measured belongs to the china holstein cows Dominant variety that Milk protein yield is high, the somatocyte scoring is low.
Wherein, the amplification system that the PCR reaction is used in the step (2) is counted with 25 μ l: 50-100ng/ μ l template DNA 1 μ l, each 1 μ l of 10pmol/ μ l primers F and R, 10mmol/LdNTP mix2.0 μ l, 5U/ μ l Taq archaeal dna polymerase 0.125 μ l, 10 * PCR reaction buffer, 2.5 μ l, surplus is distilled water;
F：5’-GGCAACTACCTGAACCAA-3’
R：5’-CACCACCCTGTCAATCAA-3’。
Wherein, the condition of PCR reaction is in the step (2): 94 ℃ 5 minutes; 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 35 seconds, 34 circulations; 72 ℃ 10 minutes.
Another object of the present invention provides described SNP and is marked at application in the china holstein cows molecular mark, and this application can be accelerated the breeding process of china holstein cows.
Described SNP is marked at the application in the china holstein cows molecular mark, may further comprise the steps:
(1) adopt polymerase chain reaction (PCR) and sequencing technologies to screen the SNP mark relevant with china holstein cows milk production trait and somatocyte scoring;
(2) detect the genotype of china holstein cows to be measured by the flight time mass spectrum method;
(3) carry out the seed selection that Milk protein yield is high, somatocyte is marked low china holstein cows Dominant variety according to genotype.
The present invention carries out gene type to the SNP site of the HAL gene of china holstein cows, and milk production trait and the somatocyte scoring of the G197A of this gene and milk cow carried out association analysis, carrying out the linear analogue analysis by SAS9.0 software Mixed process finds, the Milk protein yield of GG genotype individuality is significantly higher than AA genotype individual (P＜0.05), and the somatocyte scoring of GG genotype individuality extremely significantly is lower than AA genotype individual (P＜0.01).Detecting of this polymorphic site is for the marker assisted selection of china holstein cows milk production trait and somatocyte scoring provides scientific basis.
Detect china holstein cows HAL gene mononucleotide polymorphism, as molecule marker, the marker assisted selection of marking for china holstein cows milk production trait and somatocyte provides scientific basis with the SNP relevant with milk production trait and somatocyte scoring.
SNP mark and the application thereof relevant with china holstein cows milk production trait and somatocyte scoring of the present invention have following advantage:
(1) molecular genetic marker provided by the invention is not subjected to the restriction such as age, sex of china holstein cows, can be used for the early stage seed selection of china holstein cows, even when just birth, just can screen exactly, can significantly promote the breeding process of china holstein cows.
(2) method that detects china holstein cows HAL gene mononucleotide polymorphism accurately and reliably, and is easy and simple to handle.
(3) detecting of the SNP site of the HAL gene of china holstein cows is for the marker assisted selection of china holstein cows milk production trait and somatocyte scoring provides scientific basis.
Description of drawings
Fig. 1 is three kinds of genotype order-checking peak figure;
Wherein, (a) be the GG type; (b) be the AA type; (c) be the GA type;
Fig. 2 is three kinds of genotype flight time mass spectrum SNP somatotype cluster analysis figure;
Wherein, transverse axis region representation GG genotype is individual; Longitudinal axis region representation AA genotype is individual; Region intermediate represents that the GA genotype is individual; No call represents individual one genotype disappearance; Low Mass Height represents the lower molecular weight site; High Mass Height represents the high molecular site.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
1.1 extract the genomic dna in the china holstein cows blood to be measured
With 14 bull familys picking up from the different dairy cow farms in Beijing area the ox blood sample of totally 638 china holstein cowses be stored in-20 ℃, this ox blood sample is the ox whole blood that condenses, whole blood is divided into blood clot and serum, blood clot is dim redness, serum is clear yellow, only have white corpuscle to contain DNA in the blood, white corpuscle mainly is present in the blood clot part.
This test is adopted a day root blood DNA to extract test kit and extract genomic dna from blood clot, and concrete steps are as follows:
1) the sterilized round bottom centrifuge tube of preparation 2mL is placed a clean steel ball (diameter 5mm-6mm) in each pipe;
2) take out blood sample, after the thawing, with the surgical scissors clip approximately 200 μ l-300 μ l blood clots in centrifuge tube.Add 600 μ l cell pyrolysis liquid CL, put into tissue grinder, ground 5 minutes with 30 times/second frequencies, be placed in 55 ℃ of baking ovens and processed 2-3 minute, put upside down mixing therebetween for several times, take out steel ball and abandon;
3) 10, the centrifugal 1min of 000rpm discards the garnet supernatant;
4) again add 600 μ l cell pyrolysis liquid CL, vibrator vibration makes the throw out suspension of scattering, the centrifugal 1min of 10,000rpm, supernatant discarded;
5) add 200 μ l damping fluid GS, with the vortex instrument clot particle that fully suspends;
6) add 20 μ l Proteinase Ks, 220 μ l damping fluid GB fully rock mixing;
7) digested overnight in 55 ℃ of converters, be put upside down mixing for several times with the promoting digestion process, until the solution becomes clear is generally light brown at the digestion starting stage;
8) add the iced dehydrated alcohol of 200 μ l, the mixing that turns upside down gently, 5000 rev/mins centrifugal 2 minutes, impurity such as ox hair etc. is precipitated to the pipe end.Liquid rotating in the centrifuge tube is moved on in the adsorption column, and the centrifugal 30s of 12,000rpm discards the waste liquid in the collection tube, not centrifugal completely recentrifuge;
9) add 500 μ l protein liquid removal GD in adsorption column, left standstill 2 minutes, the centrifugal 30s of 12000rpm discards waste liquid in the collection tube;
10) add 700 μ l rinsing liquid PW in adsorption column, left standstill 2 minutes, the centrifugal 30s of 12,000rpm discards waste liquid;
11) repeat previous step;
12) 12, the centrifugal 2min of 000rpm;
13) adsorption column is transferred in the new 1.5mL centrifuge tube, opening hangs 2 minutes in 55 ℃ of baking ovens, and is complete to the ethanol volatilization;
14) the elution buffer TB of adding 100 μ l preheatings, the lid lid leaves standstill 2min in 55 ℃ of baking ovens;
15) 12, the centrifugal 2min of 000rpm abandons adsorption column.
Genomic dna in the bovine blood is present in the centrifuge tube solution, and 4 ℃ save backup or-20 ℃ of prolonged preservation.
1.2 amplification contains the nucleotide fragments in SNP site
HAL gene (ENSBTAG00000016276) primers of including according to the Ensembl database, comprise forward primer F:5 '-GGCAACTACCTGAACCAA-3 ' and reverse primer R:5 '-CACCACCCTGTCAATCAA-3 ', genomic dna in 1.1 is as template, amplify the nucleotide fragments at SNP to be measured place, shown in SEQ IDNO.1.This SNP site is positioned at the 197bp place of pcr amplified fragment, and base can be G or A herein.
Wherein, the PCR reaction system is counted with 25 μ l: 50-100ng/ μ l template DNA 1 μ l, each 1 μ l of 10pmol/ μ l primers F and R, 10mmol/L dNTP mix 2.0 μ l, 5U/ μ L TaqDNA polysaccharase 0.125 μ l, 10 * PCR reaction buffer, 2.5 μ l, surplus is distilled water.
The PCR reaction conditions is: 94 ℃ 5 minutes; 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 35 seconds, 34 circulations; 72 ℃ 10 minutes.
1.3 the detection pcr amplified fragment obtains the SNP mark
To the detection of checking order of the pcr amplification product in 1.2, if the base at 197bp place is G in the amplified production sequence, then china holstein cows to be measured belongs to the china holstein cows Dominant variety that Milk protein yield is high, the somatocyte scoring is low.Three kinds of genotypic order-checking peak figure as shown in Figure 1.
1.4 genotype is judged
Detect the genotype of china holstein cows to be measured by the flight time mass spectrum method; Result according to flight time mass spectrum judges the genotype of SNP site in detecting colony.The distributing position of signal per sample, genotype can be divided into GG type, GA type and AA type.Three kinds of genotypic somatotype results as shown in Figure 2.
1.5 above-mentioned SNP is marked at the application in the seed selection of china holstein cows Dominant variety
This SNP can be used as molecular genetic marker, seek relevant with it or the closely linked quantitative trait locus that affects Milk protein yield and somatocyte scoring, select or marker assisted selection milk cow is directly carried out genotype, thus the seed selection of quickening china holstein cows Dominant variety.
The association analysis of embodiment 2 china holstein cows different genotype and milk production trait and somatocyte scoring is used with detection
Method according to embodiment 1, to 14 bull familys picking up from the different dairy cow farms in Beijing area totally 638 china holstein cowses carry out flight time mass spectrum and detect, the analytical results in HAL gene sequence 197bp site shown in SEQ ID NO.1 is as shown in table 1.
Use SAS9.0 software Mixed program to carry out linear analogue and analyze the genotype of SNP polymorphic site and the incidence relation of milk production trait and somatocyte scoring, during analysis with individual breeding value (EBV, provided by Chinese milk cow Data centre) represent phenotypic number, the model of employing is as follows:
Y
ijk=μ+G
i+a
j+e
ijk
Wherein, Y
IjkBe individual breeding value vector, μ is breeding value colony average, G
iBe genotype effect vector, a
jBe minor-polygene effect vector, e
IjkBe the random residual effect vector.Analytical results is as shown in table 2.
Genotype frequency and the gene frequency of table 1SNP site in china holstein cows colony
As shown in Table 1, the homozygous number of individuals of GG is significantly higher than the AA type, and G allelotrope is protogene.The comptibility test of the side of card,
(1)=3.84, this site is in the Hardy-Weinberg equilibrium state in colony as can be known.
Association analysis is carried out in table 2 china holstein cows HAL gene SNP site and milk production trait and somatocyte scoring
| ? | Milk protein yield | The somatocyte scoring |
| The P value | 0.0447* | <.0001** |
| AA | 6.9854±4.7116 b | 303.23±1.7988 Aa |
| GA | 11.1322±4.3397 ab | 301.07±1.6597 Ab |
| GG | 12.5897±4.2881 a | 298.69±1.6404 B |
Numeric representation in the table 2 is least square mean value ± standard error; Letter representation difference identical in the table is not remarkable, different letter representation significant differences, capitalization represent difference extremely significantly (P＜0.01), lowercase alphabet differential different significantly (P＜0.05), * represent significant correlation (P＜0.05), * * represents utmost point significant correlation (P＜0.01).
As shown in Table 2, genotype has reached conspicuous level (P＜0.05) to the impact of Milk protein yield, and the impact that somatocyte is marked reaches utmost point conspicuous level (P＜0.01).GG genotype Milk protein yield is significantly higher than the AA type, and difference is not remarkable between GA genotype and two kinds of genotype; And the genotypic somatocyte scoring of GG extremely significantly is lower than GA type and AA type, and the GA genotype significantly is lower than the AA type.
Embodiment 3 test kits
Each composition is composed as follows in the test kit:
50-100ng/ μ l bovine blood DNA;
Primers F;
Primer R;
dNTP?mix；
5U/ μ l Taq archaeal dna polymerase;
10 * PCR reaction buffer;
Distilled water.
The amplification system that the PCR reaction is used in the test kit is counted with 25 μ l: 50-100ng/ μ l bovine blood DNA 1 μ l, each 1 μ l of 10pmol/ μ l primers F and R, 10mmol/L dNTP mix2.0 μ l, 5U/ μ l Taq archaeal dna polymerase 0.125 μ l, 10 * PCR reaction buffer, 2.5 μ l, surplus is distilled water;
F：5’-GGCAACTACCTGAACCAA-3’
R：5’-CACCACCCTGTCAATCAA-3’。
Wherein, the condition of PCR reaction is: 94 ℃ 5 minutes; 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 35 seconds, 34 circulations; 72 ℃ 10 minutes.
The reaction products therefrom is the purpose nucleotide fragments, and sequence detects the base on this sequence 197bp site shown in SEQ ID NO.1, thereby the genotype of judgement sample judges milk production trait and somatocyte scoring.
Although, above used general explanation, embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the nucleic acid fragment for detection of china holstein cows milk production trait and somatocyte scoring is characterized in that, the sequence of this nucleic acid fragment is shown in SEQ ID NO.1, and the base of 197bp site is G or A.
2. SNP mark relevant with the scoring of china holstein cows milk production trait and somatocyte, described SNP mark is positioned at the 197bp site of china holstein cows HAL gene sequence shown in SEQ ID NO.1, and the base in site is G herein.
3. for detection of the test kit of SNP mark claimed in claim 2, it is characterized in that, described test kit contains forward primer F:5 '-GGCAACTACCTGAACCAA-3 ' and reverse primer R:5 '-CACCACCCTGTCAATCAA-3 '.
4. test kit according to claim 3 is characterized in that, described test kit comprises dNTPs, Taq archaeal dna polymerase, Mg
2+With the PCR reaction buffer.
5. test kit according to claim 4 is characterized in that, described test kit also comprises standard positive template.
6. SNP claimed in claim 2 is marked at the application of identifying in the china holstein cows Dominant variety that Milk protein yield is high, the somatocyte scoring is low.
7. application according to claim 6 is characterized in that, may further comprise the steps:
(1) genomic dna of extraction china holstein cows to be measured;
(2) take the genomic dna of china holstein cows to be measured as masterplate, utilize primers F and R, amplify china holstein cows HAL gene 307bp fragment by the PCR reaction, wherein,
F:5’-GGCAACTACCTGAACCAA-3’
R:5’-CACCACCCTGTCAATCAA-3’；
(3) detect pcr amplification product, if the base at 197bp place is G in the amplified production sequence, then china holstein cows to be measured belongs to the china holstein cows Dominant variety that Milk protein yield is high, the somatocyte scoring is low.
8. application according to claim 7, it is characterized in that, the amplification system that the PCR reaction is used in the step (2) is counted with 25 μ l: 50-100ng/ μ l template DNA 1 μ l, each 1 μ l of 10pmol/ μ l primers F and R, 10mmol/L dNTP mix2.0 μ l, 5U/ μ l TaqDNA polysaccharase 0.125 μ l, 10 * PCR reaction buffer, 2.5 μ l, surplus is distilled water;
F：5’-GGCAACTACCTGAACCAA-3’
R：5’-CACCACCCTGTCAATCAA-3’；
The condition of PCR reaction is: 94 ℃ 5 minutes; 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 35 seconds, 34 circulations; 72 ℃ 10 minutes.
9. SNP claimed in claim 2 is marked at the application in the china holstein cows molecular mark.
10. application according to claim 9 is characterized in that, may further comprise the steps:
(1) adopt polymerase chain reaction and sequencing technologies to screen the SNP mark relevant with china holstein cows milk production trait and somatocyte scoring;
(2) detect the genotype of china holstein cows to be measured by the flight time mass spectrum method;
(3) carry out the seed selection that Milk protein yield is high, somatocyte is marked low china holstein cows Dominant variety according to genotype.
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